Journal article
Development, characterization and diagnostic application of a monoclonal antibody specific for a proteinase K resistant Lawsonia intracellularis antigen
Danish Institute for Food and Veterinary Research1
Section for Veterinary Diagnostics, Division of Veterinary Diagnostics and Research, National Veterinary Institute, Technical University of Denmark2
Division of Veterinary Diagnostics and Research, National Veterinary Institute, Technical University of Denmark3
National Veterinary Institute, Technical University of Denmark4
Adaptive Immunology & Parasitology, Division of Veterinary Diagnostics and Research, National Veterinary Institute, Technical University of Denmark5
Microbial Ecology, Division of Veterinary Diagnostics and Research, National Veterinary Institute, Technical University of Denmark6
Management, National Veterinary Institute, Technical University of Denmark7
Proliferative enteropathy (PE) is one of the most important infections in pigs caused by Lawsonia intracellularis, an obligate intracellular bacterium. The purpose of the present investigation was to develop monoclonal antibodies with specificity to L. intracellularis useful both for diagnostic purposes (by immunohistochemistry) and for bacterial characterization.
Several antibody producing hybridomas were established by fusion of mouse myeloma with spleen cells from BALB/c mice immunized with mucosa scrapings of the intestinal mucosa from a L. intracellularis infected pig. A monoclonal antibody (mAb), Law1-DK, isotyped as IgG2b was selected by indirect immunofluorescence antibody test (IFAT).
Histological sections of the intestines from pigs affected by proliferative enteropathy and in vitro grown bacteria in cell culture were tested positive for the presence of L. intracellularis with the mAb. A molecule at 21 kDa was recognized by the mAb in a Western blotting analysis when a whole-cell preparation of L. intracellularis was run on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
This antigen was released from L. intracellularis by mild heat treatment and was resistant to proteinase K digestion, suggesting it to be non-protein, e.g., lipopolysaccharide (LPS). This suggestion was supported by its presence in the aqueous phase of a phenol-water extract. The inhibitory effect of periodate oxidation on the antigen-antibody binding confirmed the participation of a carbohydrate epitope.
The new mAb was tested highly specific for L. intracellularis by applying in situ hybridization with a L. intracellularis specific probe targeting 16S ribosomal RNA simultaneously with the IFAT.
Language: | English |
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Year: | 2005 |
Pages: | 199-206 |
ISSN: | 18732542 and 03781135 |
Types: | Journal article |
DOI: | 10.1016/j.vetmic.2004.10.022 |
ORCIDs: | Jensen, Tim Kåre , Jungersen, Gregers and Boye, Mette |
Animals Antibodies, Monoclonal Antigens, Bacterial Desulfovibrionaceae Infections Electrophoresis, Polyacrylamide Gel Endopeptidase K Fluorescent Antibody Technique, Indirect Hybridomas In Situ Hybridization Lawsonia Bacteria Mice Mice, Inbred BALB C Phylogeny RNA, Ribosomal, 16S Swine Swine Diseases